The main goal of this research proposal is to understand how the PTH and calcitonin genes are regulated. 1,25(0H)2D3 decreased (i) PTH gene transcription in vitro in primary cultures of bovine parathyroid cells, and in vivo in the rat and (ii) calcitonin gene transcription in vivo in the rat. In vivo preliminary results show that high calcium has no effect on PTH gene transcription whilst low calcium increased transcription, in contrast to published in vitro studies. (i) It is planned to study in vivo time and dose responses of PTH and calcitonin gene transcription to acute and chronic hypercalcemia and hypocalcemia. In vitro in primary cultures of bovine parathyroid cells the effect of calcium on PTH mRNA will be separated from its effect on parathyroid cell proliferation by measuring parathyroid and fibroblast cell numbers and (3H)- thymidine uptake. (ii) The effect of 1,25(0H)2D3 in vivo will be studied: (a) priming doses given to normal rats and rats with parathyroid hyperplasia due to vitamin D deficiency or chronic renal failure, (b) the kinetics of hormone withdrawal, (c) interaction with glucocorticoids which abolishes the 1,25(0H)2D3 effect in vitro. In vitro the effect of 1,25(0H)2D3 on transcription start sites will be studied by Sl nuclease and primer extension assays. (iii) After unilateral parathyroidectomy there is marked compensatory hyperplasia of the contralateral parathyroid. c-myc and c-ras oncogenes are increased in proliferating tissue, and after cycloheximide treatment. In HL-60 cells c-myc mRNA is decreased by 1,25(0H)2D3. It is planned to measure oncogene and PTH gene transcription (a) in vivo in replicating contralateral parathyroids with or without injections of 1,25(0H)2D3 or calcium, and (b) after cycloheximide, and (c) in vitro in primary cultures of parathyroid cells where low calcium increases proliferation. In these studies the transcription rates of other genes will be studied on the same thyroparathyroid tissue viz. actin (control), calcium binding protein (specific to parathyroid), thyroglobulin (thyroid follicular cells), and calcitonin gene related peptide (CGRP) and somatostatin (specific to thyroid c-cells). These studies should help us understand the regulation of the PTH and calcitonin genes in normal and proliferating states, with relevance to both physiology and disease.